Project Summary/Abstract This mentored career development award (K08) proposal describes the research and educational plans for the candidate, Andrew Janowski MD. His long-term goal is to discover and characterize viral causes of central nervous system (CNS) infection. One entity, encephalitis, is often caused by infectious pathogens, but frequently an etiology cannot be identified despite extensive clinical testing. A subset of encephalitis cases are anticipated to be caused by novel viruses or viruses with an unappreciated neurotropism. Dr. Janowski has previously used next-generation sequencing technology to discover a novel family of viruses named statoviruses and has also increased the number of known RNA bacteriophages by over six-fold. He will apply these skills to identify novel pathogens in CNS infections and to characterize their biology. Astrovirus VA1 (VA1), a virus originally discovered by Dr. Janowski's mentor, David Wang PhD, has been identified as an emerging cause of encephalitis. However, studies of the fundamental biology of this virus has been precluded by the lack of an experimental model of infection. Dr. Janowski has developed the first cell culture model of VA1 infection in vitro, and now the biology of VA1 infection can be elucidated using this system. In Aim 1, he will develop an in vitro model of VA1 CNS infection. He will define host expression profiles upon infection and determine if there are any VA1 strain specific cellular tropisms. The host immune response to VA1 infection is also poorly defined, but preliminary evidence suggests that interferon-? is induced by VA1 and addition of exogenous interferon reduces VA1 replication. For Aim 2, Dr. Janowski will use a CRISPR/Cas- 9 approach to define pattern recognition receptors and interferon stimulated genes that mediate the interferon response. In Aim 3, Dr. Janowski will use a neutralization assay to define the seroprevalence of neutralizing antibodies to VA1. He will also use a qRT-PCR assay to define the burden of CNS diseases caused by VA1, as he has a repository of clinical specimens from patients with acute CNS diseases. Characterizing VA1 will prepare Dr. Janowski to transition as an independent physician-scientist by the end of the K08 award period. His career development will be overseen by his co-mentors, Drs. David Wang and Gregory Storch, with Drs. Robyn Klein, Herbert ?Skip? Virgin, Mike Diamond, and David Hunstad forming an illustrious advisory committee that will assist in career development and experimental design. In addition, Dr. Janowski will complete a master's degree in clinical investigation in translational medicine at Washington University in St Louis to enhance his formal scientific training. He has the support of a world-class institution, an outstanding group of mentors and advisors, a well-developed educational plan, and a research project that will provide unique and provocative results regarding the biology of VA1. The results of this K08 proposal will not only build upon host-virus interactions in CNS infections but it will launch the independent research career of Dr. Janowski.